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... these plasmids are amplified and none are lost. Each Addgene library has its own depositor transformation...counteracted by growing the cells on solid plates, where each transformant forms its own colony, uninfluenced ...straightforward – you need to count how many times each of the library components is found in the read list...counts will give you a bell curve of the abundance of each component of your library. You should look for two...’ll agree with me that they look very similar to each other - that’s exactly what we want! At the end ...

...to treat each lane with a different concentration of your reagent. Be aware that this approach requires...Alternatively, you can run a reagent gradient. In this approach, you’ll run the same protein concentration in ...from the membrane (2) and the membrane is cut so each lane is its own piece (3). After incubation, blocking...some evidence that antibodies may need longer to reach their maximum binding, so feel free to trial even.../reprobing) Check for antibody signaling after each round of stripping* Use a harsher stripping ...  Rachel Leeson...

... to make it easy for you to share plasmids. To achieve this goal, we will archive any plasmids you've ...article page, enter the name, type, and description of each plasmid associated with this deposit as explained...separate promoters, tags, or cloning information for each insert.  Encodes gRNA/shRNA A plasmid with one... plasmid has multiple inserts, enter details for each insert individually.   Figure 9: Add cloning information...information for your first plasmid and can be used on each individual page (Gene and Insert, Cloning Information...The "Copy" button is located near the top right of each data entry page. Once you have completed the data...

...huge innovation, there was a limitation to this approach. CBEs and ABEs are base transition editors. They...were included in the CGBE. They also developed a machine learning model capable of predicting editing outcomes...GBEs), Xueli Zhang’s lab, returned with a unique approach to improving C → G editing. Instead of fusing ...abasic site. In this post, it refers to the same approach as a CGBE. BER Base Excision Repair A cellular... CRISPRi screens, target-library analysis, and machine learning. Nature Biotechnology, 39(11), 1414–1425...with sequence context preference predictable by machine learning methods. Nature Communications, 12(1),...

...refresher). There are five antibody isotypes that each have a unique heavy-chain constant region: IgM, ...not all isotypes have the same ability to activate each of these functions: Recruitment of immune cells...Transportation of antibodies to places they can’t reach on their own. The antibody’s heavy-chain constant...numbered, i.e. IgG1, IgG2, etc. Let’s dive in and meet each of the isotypes. IgM This isotype is produced during...expressing IgM. IgM has a pentamer structure, which means each IgM molecule has a binding capacity or valency of...

...can be affected by both autofluorescence and photobleaching. Researchers have long been interested in using... cells. As Nano-lanterns are not subject to photobleaching/phototoxicity, they successfully imaged multiple...applications. For long-term imaging, especially where photobleaching and phototoxicity are a concern, these constructs...constructs come in very handy. This approach is especially suited to complement optogenetic tools, where...of various Nano-lanterns, but it does not isolate each Nano-lantern, so Takai et al. used a linear unmixing...

...research to study gene function. RNAi is commonly achieved by feeding E. coli expressing dsRNA to C. elegans...between two inverted T7 promoters. Transcription from each promoter progresses past the specific gene producing...TurboID and miniTurbo podcast segment A fundamental approach to studying membranes, organelles, and other subcellular...podcast segment As the primary cause for both the stomach flu and food-borne illnesses, noroviruses can infect...enriched with synbodies--or synthetic antibodies--attached to magnetic beads, allowing the virus to be easily...

...members from the Customer Service, Data Curation, Outreach, Product, Scientist, and Software teams. We knew... with an intuitive user interface.   The team approached the issue from multiple angles: the overall user...of information so we were able to fine tune our approach much more than if we were dealing with general...the strategies we used in our software design. In each case, we leveraged the team’s knowledge of the scientific...catalog, educational resources, and depositors — each with subcategories such as plasmids and viral preps...

...recombineering, bacteria express phage recombination machinery, which facilitates easy genetic modification by...methods. As described above, you design two primers, each containing the desired mutations, to amplify both...mutations, the primers also have some homology to each other; ~17 bases is optimal. After being treated...electrocompetent cells in which the phage recombination machinery is induced using L-arabinose. These cells can ...mutation-containing colonies/total colonies) is about 84% for each of the three methods. However, REPLACR-mutagenesis...

...instead use a preexisting, conserved cellular machinery to process gRNAs. Hijacking tRNA processing to...plants. Yang was intrigued by the tRNA processing machinery present in almost every living organism. Precursor...targeting 4 MAPK genes, again using rice protoplasts. Each pair of gRNAs targeted genomic sites separated by... them to examine the frequency of both indels at each locus, as well as large deletions. They detected...promoter and terminator need be encoded only once, and each <200 bp tRNA-gRNA takes up half the space of a standard...

...activate all 20,000 genes, it is not possible to test each one and make sure it’s working. You can only do ... Those two things, in my opinion, had not been achieved.” That is, until now. Enter the SAM pooled library...uses the sunCas9 system and contains 10 sgRNAs for each transcription start site in 15,977 human genes. ...repressor (CRISPRi) library contains 10 sgRNAs for each transcription start site in those 15,977 human genes...demonstrated in a paper in Cell that they could typically achieve 90-99% knockdown with minimal off-target effects...

... tested following each immunization until the desired level of antibodies is reached - typically within...immune system, antibodies are produced by B cells. Each individual B cell produces antibodies that all recognize...proteins pass through. However, protein A and protein G each have different affinities for IgG subtypes from ...experiments that require multiple antibodies that each detect a different antigen. To pre-adsorb the antibodies...

...Cell paper. With six figures. Averaging ten panels each. (For this story, it is important to know that the...to this paper and/or biology in general until I reached Fig. 6, Panel L, and hope nobody noticed that none...wrong, ask yourself, “What next step will help me achieve my purpose? Should I go over this slide again? ...the presentation, at which point you will have achieved your goal. Yay!   You’re only human When it comes... Rachel Leeson...

...foreign dsRNA activates the innate immune response machinery of the host. This pathway has been clinically... response is activated if and when dsRNA levels reach a given threshold within the cell, as is often the...viral infection. Endogenously produced dsRNA rarely reach this threshold and thus do not typically activate...and H2) handle most of these intermediates, with each one acting on specific substrates and in distinct...cycles. When RNA:DNA hybrids levels are allowed to reach very high or low levels, possibly due to dysregulation...

...came on March 9, 2005. It took almost six years to reach our first major repository milestone — 10,000 plasmids...did this the old-fashioned way: through physical outreach, visiting organizations, knocking on doors, and...approvals. In 2013 we were thrilled to receive the Massachusetts Nonprofit Network Excellence Award for our electronic...   Expanding our work In March 2015, Addgene reached the 500,000 plasmids distributed milestone! We ... Rachel Leeson...

... the target protein, so they do not compete with each other. This antibody is also conjugated to another...reflects the concentration of your target protein in each well. The reaction is stopped by the addition of...reaction, turns the solution yellow. The absorbance of each well, which is measured using a microplate reader..., so if you max out your standard curve and it reaches an asymptote, you won’t be able to accurately calculate...portion or the OD of your experimental samples has reached 4.0, you can resolve this issue when you repeat...

...thought otherwise! They suspected a data-driven approach might allow for an increase in the complexity....to sets of up to 6-8 overhangs, the data-driven approach was needed to massively expand complexity. The...search window for a site—or sites—that must be used. Each of the tools works with either 3-base or 4-base ... 10-piece circular assembly, with the 52-piece approach yielding approximately ~800-fold fewer plaques...  Rachel Leeson...

...that are easily distinguishable from each other. In a direct approach, that is simple - but what about when...a mix of different isotypes and subclasses. They each have their own advantages. Since monoclonal antibodies...use an indirect (primary and secondary antibody) approach?  Staining for multiple targets When staining ...plan primary/secondary antibody pairing such that each target has a unique species or isotype that can ...

... a good idea to prepare 1-2 unique questions for each interviewer by looking at their LinkedIn profiles.... I’d also use this notebook to take notes after each interview.   Tailor your 1-hour presentation to ... numerous people during your industry interview. Each interview is an opportunity to learn something and...clarification on the dress code for the interview. Greet each interviewer and then thank them once the interview...question. Maybe you ask for questions after completing each section of your talk. Some video conferencing software...

...number of papers with your search term published each year, which is a nice way to see growth or decline... in order to turn Twitter into a paper-finding-machine. There are four general types of accounts you can...Scholar analyzes publications and figures using machine learning to extract important features such as ...the number of papers with your key term published each year is displayed with your search results and can...your feed. Feeds are organized by papers published each day, week, or month. A measure of the number of ...